古生噬鹽紫菌Halobacterium salinarum紫色細胞膜 (purple membrane, PM) 內存在有一跨膜蛋白-細菌視紫質 (bacteriorhodopsin, BR) ，為光驅動質子泵 (light-driven proton pump)，受光後可吸收光能使質子從細胞膜內被推往細胞膜外，產生跨膜質子濃度梯度，可進而產生光電流訊號。本研究首先利用PM光電流訊號與入射光強度間成正關係及奈米金 (gold nanoparticle, AuNPs) 可遮光之事實，以固定化有單股探針基因DNA1之b-PM晶片 (ssDNA1-bPM晶片) 對不同濃度之標誌有AuNPs的單股目標基因DNA2 (ssDNA2-AuNPs) 分別進行注流式即時與靜態檢測，以光電流訊號變化差異找出最低檢測濃度，並探討AuNPs存在對檢測靈敏度之影響。在25 μL/min注流式即時檢測下，可檢測30 pM ssDNA2-AuNPs，而在靜態下檢測ssDNA2-AuNPs時比起對ssDNA2時之檢測具有有更佳靈敏度。其次利用固定化有單股引子基因DNA1'之ssDNA1'-bPM晶片對單股目標基因DNA3'進行雜合，並加入dNTP與Klenow Fragment (3' → 5' exo-) polymerase，靜態下進行基因合成之即時監測，結果發現，PM晶片可感應基因合成時所釋放之質子，而使其光電流下降。同一晶片至少可連續進行兩個鹼基之合成，證明PM晶片可應用於基因序列分析。

The purple membrane (PM) of halophilic archaeon Halobacterium salinarum contains a transmembrane protein, bacteriorhodopsin (BR), which is a light-driven proton pump capable of transporting a proton across the membrane upon illumination and subsequently generating photocurrents. Based on light scattering of gold nanoparticle (AuNPs) as well as the linear dependence of PM photocurrents on illumination intensities, a PM chip coated with single-stranded probe DNA1 was used to detect another AuNPs-conjugated single-stranded target DNA2 (ssDNA2-AuNPs) at different concentrations under both real-time flow-injection and static conditions, using PM photocurrents as the study parameter. The minimum concentration of ssDNA2-AuNPs to be detected at 25 μL/min was 30 pM, and the detection sensitivity for DNA2-AuNPs under a static condition was higher than that for ssDNA2. In addition, another PM chip coated with single-stranded primer DNA1' was used to capture single stranded target DNA3'; then the resulting PM-gene complex chip was used to real-time monitor DNA synthesis under a static condition, following the addition of a mixture containing dNTP and Klenow Fragment (3' → 5' exo-) polymerase. This complex chip sensed the protons produced during DNA synthesis and exhibited a reduction in photocurrent. At least two consecutive nucleotide incorporation cycles were detected by using a single PM-gene complex chip, demonstrating application feasibility of PM chips on gene sequencing.

Balashov SP. Protonation reactions and their coupling in bacteriorhodopsin. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2000, 1460 (1):75-94.

Cai H, Wang Y, He P, Fang Y. Electrochemical detection of DNA hybridization based on silver-enhanced gold nanoparticle label. Analytica Chimica Acta. 2002, 469 (2):165-172.

Chen HM, Jheng KR, Yu AD. Direct, label-free, selective, and sensitive microbial detection using a bacteriorhodopsin-based photoelectric immunosensor. Biosensors and Bioelectronics. 2017, 91 (1):24-31.

Chen HM, Lin CJ, Jheng KR, Kosasih A, Chang JY. Effect of graphene oxide on affinity-immobilization of purple membranes on solid supports. Colloids and Surfaces B: Biointerfaces. 2014, 116 (1):482-488.

Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chemical Reviews. 2004, 104 (1):293-346.

Duy J, Connell LB, Eck W, Collins SD, Smith RL. Preparation of surfactant-stabilized gold nanoparticle–peptide nucleic acid conjugates. Journal of Nanoparticle Research. 2010, 12 (7):2363-2369.

Eckert KA, Kunkel T. Effect of reaction pH on the fidelity and processivity of exonuclease-deficient Klenow polymerase. Journal of Biological Chemistry. 1993, 268 (18):13462-13471.

Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science. 1997, 277 (5329):1078-1081.

Fan A, Lau C, Lu J. Magnetic bead-based chemiluminescent metal immunoassay with a colloidal gold label. Analytical Chemistry. 2005, 77 (10):3238-3242.

França LTC, Carrilho E, Kist TBL. A review of DNA sequencing techniques. Quarterly Reviews of Biophysics. 2002, 35 (2):169-200.

Gao H, Qi X, Chen Y, Sun W. Electrochemical deoxyribonucleic acid biosensor based on the self-assembly film with nanogold decorated on ionic liquid modified carbon paste electrode. Analytica Chimica Acta. 2011, 704 (1):133-138.

Hampp N. Bacteriorhodopsin as a photochromic retinal protein for optical memories. Chemical Reviews. 2000, 100 (5):1755-1776.

Huang X, El-Sayed MA. Gold nanoparticles: optical properties and implementations in cancer diagnosis and photothermal therapy. Journal of Advanced Research. 2010, 1 (1):13-28.

Ion Torrent. Ion Torrent™ next-gen sequencing technology. 2014. https://www.youtube.com/watch?v=WYBzbxIfuKs&feature=youtu.be.

Illumina. Illumina sequencing technology. 2010.
https://www.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf

Illumina. An introduction of next-generation sequencing technology. 2016.
https://www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf

Jain PK, Lee KS, El-Sayed IH, El-Sayed MA. Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: applications in biological imaging and biomedicine. the Journal of Physical Chemistry B. 2006, 110 (14):7238-7248.

Kawamura I, Ohmine M, Tanabe J, Tuzi S, Saitô H, Naito A. Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2007, 1768 (12):3090-3097.

Lee K-S, El-Sayed MA. Dependence of the enhanced optical scattering efficiency relative to that of absorption for gold metal nanorods on aspect ratio, size, end-cap shape, and medium refractive index. The Journal of Physical Chemistry B. 2005, 109 (43):20331-20338.
Li F, Zhang H, Dever B, Li X-F, Le XC. Thermal stability of DNA functionalized gold nanoparticles. Bioconjugate Chemistry. 2013, 24 (11):1790-1797.

Link S, El-Sayed MA. Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods. Journal. 1999, 103 (40):8410-8426.

Merriman B, Torrent I, Rothberg JM, Team D. Progress in ion torrent semiconductor chip based sequencing. Electrophoresis. 2012, 33 (23):3397-3417.

Pourmand N, Karhanek M, Persson HH, Webb CD, Lee TH, Zahradníková A, Davis RW. Direct electrical detection of DNA synthesis. Proceedings of the National Academy of Sciences. 2006, 103 (17):6466-6470.

Roche Sequencing. 454 Sequencing Systems Workflow Technology. 2015.
https://www.youtube.com/watch?v=rsJoG-AulNE.

Saha K, Agasti SS, Kim C, Li X, Rotello VM. Gold nanoparticles in chemical and biological sensing. Chemical Reviews. 2012, 112 (5):2739-2779.

Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. Journal of Molecular Biology. 1975, 94 (3):19447-20448.

Sanger, Frederick, Steven Nicklen, and Alan R. Coulson. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences. 1977, 74 (12):5463-5467.

Shendure J, Ji H. Next-generation DNA sequencing. Nature Biotechnology. 2008, 26 (10):1135.

Wang J, Yoo SK, Song L, El-Sayed MA. Molecular mechanism of the differential photoelectric response of bacteriorhodopsin. the Journal of Physical Chemistry B. 1997, 101 (37):3420-3423.

Wang J. Vectorially oriented purple membrane: characterization by photocurrent measurement and polarized-Fourier transform infrared spectroscopy. Thin Solid Films. 2000, 379 (1):224-229.

Yguerabide J, Yguerabide EE. Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications: I. Theory. Analytical Biochemistry. 1998, 262 (2):137-156.

Yguerabide J, Yguerabide EE. Resonance light scattering particles as ultrasensitive labels for detection of analytes in a wide range of applications. Journal of Cellular Biochemistry. 2001, 84 (S37):71-81.

Zeng S, Yong K-T, Roy I, Dinh X-Q, Yu X, Luan F. A review on functionalized gold nanoparticles for biosensing applications. Plasmonics. 2011, 6 (3):491.
Zhao G, Guan Y. Polymerization behavior of Klenow fragment and Taq DNA polymerase in short primer extension reactions. Acta Biochimica Biophysica Sinica. 2010, 42 (10):722-728.

吳欣穎, 細菌視紫質泛用型免疫光電晶片製備與原子力顯微鏡分析, 國立台灣科技大學化學工程研究所碩士論文. 2015

林承德, AFM探討分別以結合氧化石墨烯之親和吸附與共價鍵結作用固定化之紫膜, 國立台灣科技大學化學工程研究所碩士論文. 2012

陳冠辰, 適體-細菌視紫質生物光電感測晶片之探討, 國立台灣科技大學化學工程研究所碩士論文. 2015

張智凱, 奈米金於細菌視紫質DNA光電晶片應用之初步探討, 國立台灣科技大學化學工程研究所碩士論文. 2012

蔡孟訓, 自排性單分子膜於bacteriorhodopsin光電晶片製備之探討, 國立台灣科技大學化學工程研究所碩士論文. 2011

廖信銓, 微流體於細菌視紫質光電晶片製備之應用, 國立台灣科技大學化學工程研究所碩士論文. 2015

鄭凱如, 細菌視紫質泛用型免疫光電晶片製備與原子力顯微鏡分析, 國立台灣科技大學化學工程研究所碩士論文. 2011